Using mutations to improve Aspergillus phytases

a technology of aspergillus and mutations, applied in the field of using mutations to improve the phytase of aspergillus, can solve the problems of false activation, low specific activity of this phytase, and no studies have successfully employed any information to improve this widely used benchmark phytase, etc., to improve the nutritional value of a food consumed by humans, increase the bioavailability of minerals, and improve the nutritional value of a food

Active Publication Date: 2007-12-18
CORNELL RES FOUNDATION INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0019]The present invention also relates to a method of in vitro hydrolysis of phytate. This method involves providing a mutant phytase of the present invention. The mutant phytase is combined with a phytate source under conditions effective to increase the bioavailability of phosphate from the phytate source.
[0020]The present invention also relates to a method of improving the nutritional value of a foodstuff consumed by humans. This method involves providing a mutant phytase according to the present invention. The mutant phytase is combined with a foodstuff consumed by humans under conditions effective to increase the bioavailability of minerals from the foodstuff. Suitable minerals can include, without limitation, iron, zinc, phosphorus, and calcium.
[0021]The present invention further relates to a method of imparting improved mineral nutritional value to a plant that is edible for consumption by animals. This method involves providing a transgene containing an isolated nucleic acid molecule of the present invention. The isolated nucleic acid molecule is operatively associated with a regulatory sequence containing transcriptional and translational regulatory elements that control expression of the isolated nucleic acid molecule in a transgenic plant cell. The method also involves providing a non-transformed plant that is edible for consumption by animals. The transgene is inserted into the genome of the non-transformed plant under conditions effective to yield a transformed plant that transgenically expresses a mutant phytase encoded by the isolated nucleic acid molecule of the present invention. The resulting transformed plant has improved mineral nutritional value compared to that of the non-transformed plant.
[0022]The mutant phytases of the present invention exhibit a number of improved attributes compared to their non-mutant counterpart phytases. For example, the mutant phytases of the present invention exhibit improved phytase activity over their non-mutant counterpart phytases. The mutant phytases of the present invention also exhibit altered pH profiles and altered pH optima that favor their use in acidic environments such as the gastrointestinal tracts of animals. The mutant phytases of the present invention exhibit such improved attributes without sacrificing their thermostability, in that the mutant phytases have equal or better thermostability than their non-mutant counterpart phytases. The mutant phytases of the present invention may also be useful to produce specific inositol phosphate metabolites or products for nutritional and biomedical applications.

Problems solved by technology

enzyme. However, it was noted in that study that the proline substitution mutant phytase displayed a tendency to aggregate and precipitate and this could have lowered its true activit
However, specific activity of this phytase is not as high as some other fungal phytases such as those produced by A. terreus or A. niger.
However, to date no studies have successfully employed any of this information to improve this widely used benchmark phytase.
Non-ruminants such as poultry and swine are unable to utilize phytate phosphorus in soy-corn based diet.
Because phytate degradation by dietary phytase takes place mainly in the stomach (Yi et al., “Sites of Phytase Activity in the Gastrointestinal Tract of Young Pigs,”Animal Feed Science Technology 61:361-368 (1996)), in which pH ranges from 2.5 to 3.5, the activity dip of PhyA at pH 3.5 really limits its efficacy in animal feeding.

Method used

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  • Using mutations to improve <i>Aspergillus </i>phytases
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  • Using mutations to improve <i>Aspergillus </i>phytases

Examples

Experimental program
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Effect test

example 1

Using Mutations to Improve Aspergillus Phytases

[0117]As used in Examples 1-13, amino acid residue 27 of the Aspergillus niger phytase corresponds to amino acid residue 50 as referenced in SEQ ID NOS:2, 6, and 12, and in the claims of the present application. Also, as used in Examples 1-13, amino acid residue 362 of the Aspergillus fumigatus phytase corresponds to amino acid residue 363 as referenced in SEQ ID NOS:4 and 10 and in the claims of the present application.

[0118]The objectives of this study included the following: (1) to compare the specific activity of mutants Q27L and Q27P in A. niger phytase as well as M362L in A. fumigatus phytase with the respective wild-type controls; and (2) to determine the impacts of these single amino acid substitutions on the pH profile and heat-tolerance of the recombinant phytases.

[0119]Site-directed mutagenesis was conducted to enhance catalytic activities of Aspergillus niger and A. fumigatus phytases. Mutation Q27L in A. niger phytase cause...

example 2

Phytase Mutations

[0120]Plasmid pYPP1 containing the cloned A. niger NRRL 3135 phyA phytase gene (Han et al., “Expression of an Aspergillus niger Phytase Gene (phyA) in Saccharomyces cerevisiae,” Appl. Environ. Microbiol. 65:1915-1918 (1999), which is hereby incorporated by reference in its entirety) was utilized to generate two mutants of Q27L and Q27P. Based on the published sequence of this phyA phytase gene (GeneBank accession no. M94550), the following oligonucleotides were synthesized to generate site specific mutations at the Gln 27 residue, corresponding to the same residue in A. fumigatus (Tomschy et al., “Optimization of the Catalytic Properties of Aspergillus fumigatus Phytase Based on the Three-Dimensional Structure,”Protein Science 9:1304-1311 (2000), which is hereby incorporated by reference in its entirety), Leu 27 5′-CTTTGGGGTCTATACGCACCG-3′ (SEQ ID NO:57) and Pro 27 5′-CTTTGGGGTCCATACGCACCG-3′ (SEQ ID NO:58). The primers were phosphorylated and the Gene Editor™ in vi...

example 3

Yeast Transformation and Protein Expression

[0122]Saccharomyces cerevisiae INVSc1 (Invitrogen) were grown in yeast extract-peptone-dextrose medium (YPD) and prepared for transformation according to the manufacturer instructions. Plasmid DNA containing Q27L or Q27P was transformed into Saccharomyces by electroporation (1.5 KV, 50 μF, 129 Ω. ECM 600 Electro Cell Manipulator, Genetronics, BTX Instrument Division, San Diego, Calif.). After incubation for 2 h at 30° C. in 1 M sorbitol without agitation, cells were plated in URA(−) selective medium to screen for positive transformants. Colonies were grown in 9 mL YPD broth for 36-48 h, and then centrifuged at 1,500 rpm, 25° C. for 10 min. The cell pellet was resuspended in YPG medium (1% Yeast extract, 2% peptone, 2% galactose) for induction of the recombinant enzyme expression. Activity in the medium was measured after 24-36 h. Transformation of plasmid DNA containing M362L in Pichia pastoris strain X33, and induction of phytase expressio...

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Abstract

The present invention invention relates to an isolated nucleic acid molecule encoding a mutant phytase and the isolated mutant phytase itself. The present invention further relates to methods of using the isolated nucleic acid molecule and the isolated mutant phytase of the present invention.

Description

[0001]This application claims the benefit of U.S. Provisional Patent Application Serial No. 60 / 410,736, filed Sep. 13, 2002, which is hereby incorporated by reference in its entirety.[0002]The subject matter of this application was made with support from the United States Government under USDA Project No. 6435-13410-002-00D. The U.S. Government may have certain rights.FIELD OF THE INVENTION[0003]The present invention relates to using mutations to improve phytases of Aspergillus. BACKGROUND OF THE INVENTION[0004]Phytase enzymes are a group of histidine acid phosphatases with great potential for improving mineral nutrition and protecting the environment from phosphorus pollution coming from animal waste (Lei et al., J. Appl. Anim. Res. 17:97-112 (2000)). Aspergillus niger NRRL 3135 phyA phytase has been cloned (Mullaney et al., “Positive Identification of a Lambda gt11 Clone Containing a Region of Fungal Phytase Gene by Immunoprobe and Sequence Verification,”Appl. Microbiol. Biotechno...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): A23L1/28A01N63/00C12N5/04C12N9/16A23K1/165A23L5/20A23L11/30A23L29/00
CPCA23K1/1653A23K1/1656A23L1/0153C12Y301/03026A23L1/2113C12N9/16C12Y301/03008A23L1/034A23K20/189A23K10/14A23L5/25A23L29/06A23L11/33
Inventor LEI, XINGENMULLANEY, EDWARD J.ULLAH, ABUL H. J.
Owner CORNELL RES FOUNDATION INC
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